Measurement of light energy is becoming a very attractive method for monitoring the presence or concentration of substances in various media. Numerous bioluminescent and chemiluminescent reaction systems have been devised (Schroeder, et al., Methods in Enzymology, Vol. LVII: 424-462 (1978); Zeigler, M.M., and T.O. Baldwin, Current Topics In Bioenergetics, D. Rao Sanadi ed., (Academic Press):65-113 (1981); DeLuca, M., Non-Radiometric Assays: Technology and Application in Polypeptide and Steroid Hormone Detection, (Alan R. Liss, Inc.):47-60 and 61-77 (1988); DeJong, G.J., and P.J.M. Kwakman, J. of Chromatography, 492:319-343 (1989); McCapra, F. et al., J. Biolumin. Chemilumin., 4:51-58 (1989); Diamandis, E.P., Clin. Biochem., 23:437-443 (1990); Gillevet, P.M., Nature, 348:657-658 (13 Dec., 1990); Kricka, L.J., Amer. Clin Lab., Nov/Dec:30-32 (1990)).
Use of the luminescent molecules lucigenin, acridan dyes and their acridinium derivatives in chemiluminescent reactions and in the development of nonisotopic ligand binding assays has been extensively reported and reviewed (Weeks, I. et al., Clin. Chem. 29/8:1474-1479 (1983); Weeks, I. and J.S. Woodhead, Trends in Anal. Chem. 7/2:55-58 (1988)). The very short lived emission of photons (&lt;5 sec) to produce the flash-type kinetics in the presence of H.sub.2 O.sub.2 and NaOH oxidation reagents (pH 13.0) is characteristic of the system.
Luminescence is the production of light by any means, including photoexcitation or a chemical reaction. Chemiluminescence is the emission of light only by means of a chemical reaction. It can be further defined as the emission of light during the reversion to the ground state of electronically excited products of chemical reactions (Woodhead, J.S. et al., Complementary Immunoassays, W.P. Collins ed., (John Wiley & Sons Ltd.), 181-191 (1988)). Chemiluminescent reactions can be divided into enzyme-mediated and nonenzymatic reactions. It has been known for some time that the luminescent reactant luminol can be oxidized in neutral to alkaline conditions (pH 7.0-10.2) in the presence of oxidoreductase enzymes (horseradish peroxidase, xanthine oxidase, glucose oxidase), H.sub.2 O.sub.2, certain inorganic metal ion catalysts or molecules (iron, manganese, copper, zinc), and chelating agents, and that this oxidation leads to the production of an excited intermediate (3-aminophthalic acid) which emits light on decay to its ground state, (Schroeder, H.R. et al., Anal. Chem. 48:1933-1937 (1976); Simpson, J.S.A. et al., Nature 279:646-647 (14 June, 1979); Baret, A., U.S. Pat. No. 4,933,276)). Other specific molecules and derivatives used to produce luminescence include cyclic diacyl hydrazides other than luminol (e.g., isoluminols), dioxetane derivatives, acridinium derivatives and peroxyoxylates (Messeri, G. et al., J. Biolum. Chemilum 4:154-158 (1989); Schaap, A.P. et al., Tetrahedron Lett. 28:935-938 (1987); Givens, R.S. et al. ACS Symposium Series 383; Luminescence Applications, M.C. Goldberg ed., (Amer. Chem. Soc., Wash. D.C.: 127-154 (1989)). Additional molecules which produce light and have been utilized in the ultrasensitive measurement of molecules are polycyclic and reduced nitropolycyclic aromatic hydrocarbons, polycyclic aromatic amines, fluorescamine-labeled catecholamines, and other fluorescent derivatizing agents such as the coumarins, ninhydrins, o-phthalaldehydes, 7-fluoro-4- nitrobenz-2,1,3-oxadiazoles, naphthalene-2,3-dicarboxaldehydes, cyanobenz[f]isoindoles and dansyl chlorides (Simons, S.S., Jr. and D.F. Johnson, J. Am. Chem. Soc. 98:7098-7099 (1976); Roth, M., Anal, Chem. 43:880-882 (1971); Dunges, W., ibid, 49:442-445 (1977); Hill, D.W. et al., ibid, 51:1338-1341 (1979); Lindroth, P. and K. Mopper, ibid, 51:1667-1674 (1979); Sigvardson, K.W. and J.W. Birks, ibid, 55:432-435 (1983); Sigvardson, K.W. et al., ibid, 56:1096-1102 (1984); de Montigny, P. et al., ibid, 59:1096-1101 (1987); Grayeski, M.L. and J.K. DeVasto, ibid, 59:1203-1206 (1987); Rubinstein, M. et al., Anal. Biochem. 95:117-121 (1979); Kobayashi, S.-I., et al., ibid, 112:99-104 (1981); Watanabe, Y. and K. Imai, ibid, 116:471-472 (1981); Tsuchiya, H., J. Chromatoq. 231:247-254 (1982); DeJong, C. et al., ibid, 241:345-359 (1982); Miyaguchi, K. et al., ibid, 303:173-176 (1984); Sigvardson, K.W. and J.W. Birks, ibid, 316:507-518 (1984); Benson, J.R. and P.E. Hare, Proc. Nat. Acad. Sci. 72:619-622 (1975); Kawasaki, T. et al., Biomed. Chromatog. 4:113-118 (1990)).
There are currently three known nonenzymatic systems: the acridinium derivatives (McCapra et al., British Patent No. 1,461,877; Wolf-Rogers J. et al., J. Immunol. Methods 133:191-198 (1990)); isoluminols and the metalloporphyrins (Forgione et al., U.S. Pat. No. 4,375,972)). These systems have certain advantages over the enzyme-mediated systems in that they have faster kinetics resulting in peak light output within seconds. The metalloporphyrins are small hapten molecules which decrease stearic hinderance problems in antigen binding. In addition, the only tetrapyrrole molecules previously known to be luminescent are those containing a paramagnetic metal ion with emission yields above 10.sup.-4 (Gouterman, M., The Porphyrins, Vol. III, Dolphin, D., ed., (Academic Press): 48-50, 78-87, 115-117, 154-155 (1978); Canters, G.W and J.H. Van Der Waals, ibid,: 577-578). It has also been known that metalloporphyrins, hyposporphyrins, pseudonormal metalloporphyrins and metalloporphyrin-like molecules such as metallic chlorins, hemes, cytochromes, chlorophylis, lanthanides and actinides undergo oxidation/reduction reactions which are either primary or secondary to structural perturbations occurring in the metallic center of these molecules and that their reactive ability to catalyze the production of chemiluminescence has been ascribed to the metallo center of these molecules (Eastwood, D. and M. Gouterman, J. Mol. Spectros. 35:359-375 (1970); Fleischer, E.B. and M. Krishnamurthy, Annals N.Y. Academy of Sci. 206:32-47 (1973); Dolphin, D. et al., ibid, 206:177`201; Tsutsui, M. and T.S. Srivastava, ibid, 206:404-408; Kadish, K.M. and D.G. Davis, ibid, 206:495-504; Felton, R.H. et al., ibid, 206:504-516; Whitten, D.G. et al., ibid, 206:516-533; Wasser, P.K.W. and J.-H. Fuhrhop, ibid, 206:533-549; Forgione et al., U.S. Pat. No. 4,375,972; Reszka, K. and R.C. Sealy, Photochemistry and Photobiology 39:293-299 (1984); Gonsalves, A.M.d'A. R. et al., Tetrahedron Lett. 32:1355-1358 (1991)). These reactions are altered by iron and other metal ions which may be present in the reactants and these metal ions can interfere with and greatly confound the assay of metalloporphyrin conjugate concentrations (Ewetz, L. and A. Thore, Anal. Biochem. 71:564-570 (1976)). Different metals will strongly influence the lifetimes and luminescent properties of the metalloporphyrins.
The following metalloporphyrins will fluoresce: Zn(II), Sn (IV) , Mg(II) , Pb(II) , A1, Cd, Si(IV) , Ge(IV) , Ba, Sr, Be, Sc(III), Ti(IV), Zr(IV) , Hf(IV) , Nb(V), Ta(V), Pd(II) and Pt(II). Of these, only Zn(II) could conceivably be incorporated into a nonmetallic porphyrin in aqueous solution at a significant rate, and none would incorporate under strongly basic conditions (personal communication: Jerry C. Bommer, Ph.D.; Porphyrin Products, Logan, UT). Non-fluorescent metalloporphyrins at room temperature are: Fe, Ni, Vo, Ru, Cu, Ag, Co, Rh, and Ir (Porphyrins and Metalloporphyrins, Smith, K.M., ed., (Elsevier)).
Metalloporphyrins and nonmetallic porphyrins have both long wavelength electronic transitions and relatively long-lived excited states which make them ideal photosensitizing reagents (Hopf, F.R. and D.G. Whitten, The Porphyrins, Vol. II, Dolphin, D., ed., (Academic Press):162 (1978)). The photoexcited states of the porphyrins are pi, pi, states associated with the porphyrin macrocycle and this photoexcitation can lead to fluorescence and/or phosphorescence (Weiss, C.H. et al., J. Mol. Spectros. 16:415-450 (1965); Eastwood, D. and M. Gouterman, J. Mol. Spectros. 35:359-375 (1970)). In the case of photoexcitation of porphyrins in general, the sensitizer porphyrin molecule is raised to a short-lived singlet excited state by the absorption of a photon. This singlet state spontaneously converts to an excited triplet state capable of abstracting an electron from adjacent molecules to yield the oxidized high energy intermediate, which on decay to the ground state may emit a photon. The excited triplet state can also transfer excitation energy to ground state molecular oxygen to produce extremely reactive singlet oxygen species, also capable of oxidizing neighboring molecules to their activated intermediates (Spikes, J.D., Primary Photo-Processes in Biology and Medicine, Benasson, R.V. et al., eds. (Plenum Pub. Corp.) 209-227 (1985)). It is also known that cobalt, palladium, and, to a lesser extent zinc metalloporphyrins and all monomeric nonmetallic porphyrins are capable of generating high quantum yields of singlet oxygen (0.1-0.2 quanta) in the presence of light and molecular oxygen in solution and when polymer bound (Salokhiddinov, K.I. et al., Chem Phys Lett 76:85-87 (1980); Pashchenko, D.I. et al., Akademiia Nauk SSSR 265:889-892 (1982); Byteva, I.M. and G.P. Gurinovich, Opt, Spectrosc (USSR) 62:560-561 (1987); Nonell, S et al., Photochem. Photobio. 53:185-193 (1991)).
Electron transfer and associated chemical reactions of nonmetallic porphyrins in purified aprotic media (dimethylformamide, (DMF)) have been studied using electrochemistry (Wilson, G.S. and B.P. Neri, Annals N.Y. Academy of Sci. 206:568-578 (1973)). Polarographic and cyclic voltammetry measurements both demonstrate a two step reversible one-electron reduction of the porphyrin ring followed by a third irreversible two-electron step. Nonmetallic tetrapyrrole porphyrins have also been shown to undergo 2-4 one electron reductions and 2 one electron oxidations. The first reduction product has been shown to be a pi-anion radical (Felton, R.H. and H. Linschitz, J. Am. Chem. Soc. 88:1113-1116 (1966)), and the first oxidation a pi-cation radical (Wolberg, A. and J. Manassen, J. Am. Chem. Soc. 92:2982-2991 (1970)) by visible and electron spin resonance spectra. The second reduction and oxidation products are the dianion and dication. When the reductions of tetraphenylporphyrin and deuteroporphyrin were studied in more detail, (Dolphin, D. et al., J. Am. Chem. Soc. 92:743-745 (1970); Peychal-Heiling, G. and G.S. Wilson, Anal. Chem. 43:550-556 (1971 )) it was found that the third and fourth reductions were quite complex. Disproportionation occasionally occurred, but the highly nucleophilic materials more often abstracted protons from the solvent.
The redox cycling capability of 5-(4-nitrophenyl)-penta-2,4-dienal (NPPD) to stimulate oxygen uptake and induce superoxide anion and hydrogen peroxide formation in an NADPH-supported enzymatic system has been reported (Docampo, R. et al., Chem.-Biol. Interactions (Elsevier Scientific Publishers Ireland Ltd.) 65:123-131 (1988)).
The ability of the enzyme ferredoxin oxidoreductase to generate superoxide, hydrogen peroxide and hydroxyl free radicals from molecular oxygen in the presence of its substrate ferredoxin has been reported (Misra, H.P. and I. Fridovich, J. Biol. Chem. 246:6886-6890 (1971); Allen, J.F., Biochem. and Biophys. Res. Comm. 66:36-43 (1975); Hosein, B. and G. Palmer, Biochim. et Biophys. Acta 723:383-390 (1983); Youngman, R.J. et al., Oxy Radicals and Their Scavenger Systems, Vol II, Greenwald, R.A. and G. Cohen, eds., (Elsevier Science Pub. Co., Inc.) 212-217 (1983); Bowyer, J.R. and P. Camilleri, Biochim. et Biophys. Acta 808:235-242 (1985); Morehouse, K.M. and R.P. Mason, J. Biol. Chem. 263:1204-1211 (1988)). This enzyme produces species which have the possibility of mediating the oxidation of luminol which will in turn lead to the production of measurable photons as in the xanthine oxidase/hypoxanthine system patented by Baret, supra. This ferredoxin oxidoreductase system has not, however, been reported on as one which can be used to mediate chemiluminescence.
An augmentation of hydroxyl radical generation in the horseradish peroxidase/H.sub.2 O.sub.2 /NADPH/Fe3.sup.+ -EDTA mixture has been attributed to the addition of uroporphyrin I, haematoporphyrin and haematoporphyrin derivative (Van Steveninck, J. et al., Biochem. J. 250:197-201 (1988)). These authors have also demonstrated that rat hepatic microsomal oxidoreductases in the presence of NADPH and uroporphyrin I will generate a porphyrin anion free radical under anaerobic conditions. Aerobic incubations demonstrated the reduction of oxygen to superoxide, but, no oxygen consumption above basal levels could be detected (Morehouse, K.M et al., Archives of Biochem. and Biophys. 257:276-284 (1987)).
The use of luminescent reactions at the surface of light conductive materials (e.g., fiber-optic bundle) is the basis of the development of luminescent sensors or probes (Blum, L.J. et al., Anal. Lett. 21:717-726 (1988)). This luminescence may be modulated by specific protein binding (antibody) and can be produced in a microenvironment at the surface of the probe. The light output is then measured by photon measuring devices in the formulation of homogeneous (separation free) assays (Messeri, G. et al., Clin. Chem. 30:653-657 (1984); Sutherland, R.M. et al., Complementary Immunoassays, Collins, W.P., ed., (John Wiley & Sons, Ltd.) :241-261 (1988)).
It has been demonstrated that charged synthetic polymers (poly-N-ethyl-4-vinylpyridinium bromide, PEVP) can completely inhibit the production of light by charged conjugate molecules through electrostatic interactions. This has particularly been studied in the enhanced luminol chemiluminescent reaction catalyzed by the negatively charged peroxidase enzyme. Addition of low-molecular-weight electrolytes will eliminate this inhibition thereby supporting an electrostatic nature of the observed effect (Valsenko, S.B. et al.,J. Biolum. Chemilum. 4:164-176 (1989)).
Luminescent capillary electrophoresis gels, gel transfers or blots (Southern, Western, Northern and Dot) are examples of techniques which provide quantitative measurement of proteins and nucleic acid genetic material. These techniques can be used in conjunction with methods which amplify analyte expression, e.g., probes, PCR (polymerase chain reaction) bands, RFLP (restriction fragment length polymorphisms) methods and other methods which amplify gene expression and other analytes (Stevenson, R., Biotech. Lab. 8:4-6 (1990)).
A nonmetallic tetrapyrrole as described herein is a tetrapyrrole which has no associated central paramagnetic metal ion.
No ability to chemiluminesce or catalyze or mediate chemiluminescence has ever been attributed to any of the nonmetallic tetrapyrroles including the nonmetallic tetrapyrrole porphyrins or nonmetallic tetrapyrrole porphyrin-like molecules such as the phlorins, porphycenes, secophyrins, texaphyrins and nonmetallic chlorins. For purposes of efficiency as used herein, unless otherwise indicated, the terms "nonmetallic tetrapyrrole" or "nonmetallic tetrapyrrole porphyrin" include nonmetallic tetrapyrrole porphyrin-like molecules.
The use of a novel substance such as a nonmetallic tetrapyrrole to mediate chemiluminescence and/or act as a chemiluminescent label would be extremely beneficial because of the small size of these molecules which would minimize steric hinderance of specific binding protein (such as antibody) to labeled analyte. This would facilitate the ability of the nonmetallic tetrapyrroles to continue to function as chemiluminescent labels following binding to analytes. These nonmetallic labels are not subject to perturbations in activity brought on by the presence of varying types and amounts of heavy metals in samples.
Also it would be beneficial in improving assay sensitivity to increase the output of light obtained from a chemiluminescent reaction by improving existing signal solutions and to have novel signal solutions which provide a greater intensity of light during chemiluminescent reactions. The ability to modulate the kinetics of light output through manipulation of the signal solution formula is particularly beneficial in tailoring assays for a variety of uses (genetic probe, sensor, hormones, etc.).
Nor is there a known chemiluminescent assay for detecting multiple analytes in the same sample. Such an assay would be extremely useful in the area of chemical and medical diagnostics.